Redirected T cell therapies Flashcards
1
Q
How did cellular immunotherapy start?
A
- Cellular immunotherapy really started as HSC transplantation for the treatment of pts with blood cancers
o Patient with leukaemia given chemo with aim of eliminating any residual leukaemia – also eradicates residual haematopoiesis
o Taking cells from a HLA-matched donor and inject into bone marrow into pt with refractory acute leukaemia
o In the 60s we thought this worked as we were removing diseases BM and replacing it with healthy BM
Kolb experiment here - basically white cells lead to CML remission
2
Q
Kolb et al (1990)
A
observed that in the pts with CML that relapsed after transplanting you could go back to the original donors and ask them to donate white cells. Infusing these white cells would turn a significant proportion of these patients back into remission
3
Q
Draw scFV and CAR T cell
A
4
Q
What is scFV?
A
- This is a fusion protein composed of the variable regions of heavy and light chains of monoclonal antibodies, connect via a serine-glycine linker sequence
5
Q
Structure of CAR T cell
A
CAR T cells consist of one scFv, linked via a spacer and transmembrane domain to intracellular molecules which elicit effector functions of the T cell which they are part of
6
Q
Eshhar et al 1993
A
- construction of first-gen CAR T cells – consist of intracellular domain of CD3z, responsible for ‘signal 1’ of T cell activation
7
Q
Maher et al 2002
A
- Following the construction and antigen stimulation of CD28-CD3z dual-signalling receptors, it was found that these were associated with increased IL-2 secretion as well as increased T cell proliferation with repeated antigen exposure in the absence of any exogenous stimulation, when compared with controls with receptors only expressing CD3z or only CD28
8
Q
What are other T cell costimulatory domains being investigated?
A
4-1BB (CD137), OX40 (CD134), DAP10 and ICOS
9
Q
Discuss manufacture of CAR T cells
A
- Manufacture ex vivo genetic manipulation of autologous T cells
- Do leukapheresis from a patient’s blood activate and modify the T cells to express a transgene encoding a tumour specific CAR, infuse CAR T back into patient after chemotherapy
- Gene transfer through retroviral or lentiviral vectors
10
Q
Sommermeyer et al 2016
A
- demonstrated in a preclinical model of Raji tumour-bearing immunodeficient mice receiving CAR T cells of distinct T cell subtypes. They found that there was synergistic antitumour activity of CD4+ and CD8+ T cell subtypes in a 1:1 ratio, compared to reduced antitumour activity when either CD8+ or CD4+ were omitted from the formulated product, shown by decreased survival and increased tumour growth seen via bioluminescence imaging
11
Q
Xu et al 2014
A
- analysis of 14 patients with B cell malignancies that had been infused with autologous CD19-redirected CAR T cells. They found that the expansion of these cells in vivo correlated with the frequency of TSCM cells infused in the first 6 weeks after infusion
- It is thought that naïve T cells (TN) differentiate to form TSCM and TCM cells that are capable of self-renewal, and that these provide populations of TEM and TEFF cells that are incapable of self-renewal, suggesting that the longevity of less differentiated cells provide a greater therapeutic efficacy
12
Q
Discuss mechanisms of CAR T killing/cytotoxicity
A
- CAR T cells are thought to facilitate their effects through the perforin and granzyme axis, the Fas and Fas ligand (FasL) axis, as well as the release of cytokines
- When EGTA is used to block perforin release, most CAR T cell-mediated killing is abrogated
- Has also been shown that ectopic Fas expression on tumour cells is able to improve CAR T cell activity
- To expand further, a secondary role of cytokines has been implicated in both types of therapy. This is thought to play a role in ‘bystander-lysis’, where cytokines that are released following activation of the redirected T cell cause lysis of antigen-negative tumour cells in close proximity to the antigen-positive tumour cells engaged, a process implicated in both BiTEs and CAR T cells
- Capable of serial killing, whereby one T cells is activated and kills several targets
13
Q
What is CD19 normally? Which malignancies is it involved in and on what proportion of these is it expressed?
A
- CD19 is a transmembrane glycoprotein required for normal B cell development
- It is expressed on 95% of haematological malignancies including B-ALL, chronic lymphocytic leukaemia and B cell non-Hodgkin’s lymphoma
14
Q
Describe B-ALL
A
- Most common in children and young adults and prior to such CAR T cell therapy, it had a median survival time of 6 months in adults, poor prognosis
15
Q
Levin 2014
A
- Previously classically tested on transplanted tumours - does not mimic TME
- Compared the tumour response of erbB2-specific CAR T cells in the Her2NG transgenic mouse – overexpresses human erbB-2 transgene under MMTV promotor – these mice progressively develop mammary tumours
- They found a single administration of CAR T cells lead to rejection of the primary tumour, but a few weeks later tumours relapses in mammary gland and remote sites. This could be controlled by repeated CAR T cell injections and one mice remained cancer free for 500 days following treatment
- They also found that CAR T cells could be used prophylactically a single injection of CAR T cells before the appearance of any mammary tumours could significantly delay the appearance of mammary tumours for several months
- Could we use them in high-risk patients prophylactically? – probably not (SEs)
16
Q
Dr Rosenberg’s group (2014)
A
The first group to report responses in human B cell lymphomas and leukaemias
Describe the outcome of phase I/II study in pts with advanced B cell malignancies using CD19-specific autologous CAR T cells resulted in 8/13 patients with complete remission
17
Q
Discuss Kymirah
A
- Kymriah was approved for patients aged 25 or younger with B cell acute lymphoblastic leukaemia (ALL), following success in clinical trials.
18
Q
ELARA trial
A
- The ELARA trial of 52 patients with follicular lymphoma showed an overall remission rate of 83% within three months of treatment with Kymirah. However, in this trial, 49% of patients developed cytokine release syndrome (CRS), an adverse side effect consisting of a systemic inflammatory response. This side effect has been responsible for several deaths in similar trials.
- For example, one study was terminated following death of 5 patients as a result of fulminant cerebral oedema.
19
Q
Describe Yescarta - what is it used for?
A
- Yescarta was the second CD19 CAR T cell therapy approved in 2017 by the FDA for adults with relapsed/refractory large B cell lymphoma.
20
Q
How are Kymirah and Yescarta administered?
A
- Both therapies are administered as a one-time infusion and require lymphodepleting preconditioning prior to infusion to create a favourable environment for CAR T cells, namely through the elimination of regulatory T cells
21
Q
How does CRS present? What is given to reduce it?
A
Fever
Fatigue
Anorexia
Hypotension
Tachycardia
anti-IL-6 receptor Ab or corticosteroids
22
Q
How does CAR T/BiTE neurotoxicity present?
A
- This neurotoxicity typically presents as confusion and delirium, although seizures and the aforementioned cerebral oedema may also occur
23
Q
Avery Posey October 2020
A
- showed, via multiple independent single-cell RNA sequencing databases and subsequent bioinformatic analysis, that mural cells, which have a critical role in blood-brain-barrier (BBB) integrity, express CD19!
- They confirmed this by performing IHC with an anti-human CD19 antibody in healthy subjects post-mortem; CD19 expression was found in cells adjacent to vessel basement membrane walls in perivascular area, along both smaller capillaries and larger vessels.
- They also found that the expression of CD19 in human cells is significantly higher than in murine mural cells, implicating possible limitations of mouse models to study immunotherapy-associated toxicity.
- Therefore, a cause of this neurotoxicity may be the targeting of CD19+ mural cells and subsequent BBB disruption.
- However, there are further points which this study might have expanded on; the group have not demonstrated that CAR T cell or BiTE targetting of CD19 in human mural cells is the direct cause of clinical neurotoxicity, and contribution of other processes, such as CRS, may also be important to the pathology.
24
Q
Roybal 2016
A
- constructed a T cell expressing a combinatorial circuit using a synthetic Notch (SynNotch) receptor, which, following target antigen binding, leads to cleavage of the receptor and release of a transcriptional activator.
- This inducible transcription factor (TF) is able to regulate transcription of a CAR recognising a second tumour antigen.
- Thus, this AND-gate circuit requires binding of antigen to both the SynNotch receptor and the CAR for subsequent T cell activation
- However, they still have little control over these cells after adoptive transfer
- A key observation in this study was their in vivo work; they used mice with a CD19 K562 tumour on one flank and a GFP+/CD19+ K562 tumour on the other.
- They injected T cells bearing an a-GFP synNotch regulating a CD19 CAR and monitored tumour growth.
- They found that in all animals, these cleared the dual-antigen GFP/CD19 tumours but did not clear the CD19+-only tumours which grew at similar rates to negative controls.
- This showed that these T cells would only activate and kill targets in response to multiple tumour antigens, a potential mechanism to spare ‘bystander’ cells expressing single antigens, limiting associated on-target toxicity, such as B cell aplasia or neurotoxicity.
- The study by Posey’s group builds upon this, identifying several genes, including CDJ4,* *HLA-DRA* and *LTP, which are highly enriched on B cells in comparison to brain mural cells which serve as potential targets for combinatorial recognition.
- A limitation of an approach like this may be that both antigens would have to be expressed at considerably high levels by tumour cells.
- Also found that the CAR stays on the surface of the cell for 8 hours – their model showed no cross-reactivity so maybe it is long enough
25
Draw Roybals combinatorial antigens
26
Describe NOT gates
* An example is where PD-1 and CTLA-4-based inhibitory CARs (where the antigen scFv is fused to the signalling domains of these inhibitory receptors) are able to recognise non-specific target proteins and attenuate T cell activation.
* Similarly, **Posey’s group** found genes more enriched in mural cells than B cells, such and *BGN**,* *FN1* and *SEMA5A**,* which could serve as future targets in the prevention of neurotoxicity via NOT gates
27
Juillerat et al. (2017)
showed that the ability of these HIF-CAR T cells to kill target cells was significantly improved in hypoxic compared to normoxic conditions; although a caveat of this may be off-target effects in healthy hypoxic tissues, such as the bone marrow.
28
Huang 2020
* Current challenge is predicting optimal number of cells to infusion – need better ctrl in space/time
* Off-switches permanently down-regulate activity of CAR T cells
* Developed on-switch CARs which can be activated by light in a confined space
* Cre-loxP expression of CD19-CAR induced by blue light via nuclear translocation and dimerisation of CRY2/CIB1 pair.
* Tested in Jurkat cell line (immortalised T cell line) and primary human T cells, subcutaneous injection into mice - saw long lasting spatial controlled action
* Address duration of light dosing regimens (24hrs in study) + how long CAR activity continued after light stimulus removal
* Blue light doesn’t penetrate tissue as well as red – further develop a system using red, or implantable LEDs
* Induction ability only lasted two days
29
Mechanisms of resitance/relapse to CAR T
Loss of CD19 (2/3 of cases of relapse in ALL are due to this). Could generatee CAR with multiple antigens. CAR T cells taregtting CD20 and CD22 are in development. Another approach is to increase antigen expression on the target cells small molecule gamma secretase inhibitors have been shown to increase B cell maturation protein (BCMA) expression on the surface of myeloma cells by impairing the cleavage of surface expressed BCMA. The combination treatment with BCMA-targeted CAR T cells is now in clinical trial
CAR T transduction of the tumour. Give a tumour cell the CAR T. Masking. Only been described in a few cases.
Trogocytosis. CAR strips Ag off the tumour and allows them to evade. Mainly in preclinical models, not sure how relevant it is.
T cells dont work - they get overstimulated and exhausted - Equyem 2017 and Liu 2021
Evasion of appoptosis
Immunosuppressive TME- recruiing Tregs for example
30
* **Eyquem et al (2017)**
* TRAC T cells
* Most current approach to making CAR T cells insert the CAR-encoding gene into the T cells without disrupting the resident TCR gene
* TRAC T cells – TCR alpha chain constant region
* Used a CRISPR-Cas9 gene editing approach.
* Authors introduced RNA encoding Cas9 into T cells as well as gRNA that targets the TRAC sequence – ds break in TRAC and a viral vectore with a CAR sequence flanked by sequences homologous to the TRAC sequence was introduced into the T cells – original TRAC sequence replaced by CAR sequences
* Deleted the endogenous TCR and replace it with a CAR TCR which was under the control of the promoter for the TCR. Expression regulated in the normal way of a TCR, avoiding overactivation of the T cell.
* They showed this avoided exhaustion in vivo. Used a B-ALL mouse model. Used FACs to determine the number of cells expressing exhaustion markers on day 17 following induction. Found conventional CAR T cells showed up to 50% positive expression for three markers of exhaustion by day 17 whereas less than 2% of the TRAC-CAR T cells did
* Also showed a marked improvement in survival in the mouse model
* Could the integration of viral vectors randomly in the genome be a cause of cancer?
* **Idea for future exp** **** **compare how KOs of components of T cell exhaustion pathways compare with this CAR integration**
31
Liu et al (2021)
* generation of a switch receptor
* Generated a new chimeric antigen construct in which they linked the PD-1 EC domain to an IC activating domain of CD28 – instead of PD-1 transducing a negative signal, PD-1 transduces an activating signal
* Found that CD19-PD-1/CD28-CAR T cells had superior T-cell proliferation, cytokine production, and sequentially capability of killing PD-L1+ B-cell lymphoma cells *in vitro* and *in vivo* relative to the prototype, CD19-CAR T cells.
32
Reinhard et al. (2020)
* designed a CAR targetting tight junction protein claudin-6 (CLDN6), a human tumour-specific antigen.
* They designed a nanoparticulate RNA vaccine, CLDN6-LPX, which promoted CLDN6 expression on the surface of dendritic cells.
* They used mice bearing CLDN6+ lung tumours and administered a subtherapeutic dose of mouse CLDN6 CAR T cells followed by CLDN6-LPX or control.
* It was found that, in the mice receiving CLDN6 CAR T cells and control, tumour growth was only delayed, but the mice receiving CLDN6-LPX demonstrated complete tumour regression and higher median survival.
* Therefore, RNA vaccines could be used clinically to promote CAR T cell expansion for treatment of solid tumours.
33
Describe how 'off-the-shelf' CAR T cell therapy would work
* ‘Off the shelf therapy’ Take a healthy donor T cell population, ex vivo editing to KO the HLA which enables the T cell to be accepted in multiple populations without being rejected. You KO the TCR and then insert CAR T gene.
34
Qasim, 2017
* Proof-of-concept evidence for allogeneic therapy
* 2 paediatric patients with B-ALL
* Used TALENs to engineer HLA-mismatched donor T cells
* Donor T cells were transduced with lentivirus to express CAR-CD19 and these were electroporated with TALENs to ablate CD52 and T cell receptor- alpha constant region
* CD52 so the cells could evade the conditioning therapy before transplantation and TCR-alpha constant region to minimise graft-vs-host disease
* Molecular remission seen within 1 month in both infants (1 had evidence of skin GVHD, managed with steroids)
* Saw long term remission
35
When was the first BiTE manufactured? Describe
* First BiTE was manufactured in 1995, targeting 17-1A, a tumour antigen, with one ScFv arm and CD3 with the other
36
Describe and draw structure of BiTEs and discuss their manufacture
* Recombinant soluble protein, consisting of two different ScFvs, usually one targeting CD3 on T cells and the other directed at tumour surface antigens, again, joined by a short flexible linker Fig 2
* Manufacture BiTEs can be secreted from mammalian cell lines and bacteria as recombinant single chain polypeptides in large quantities
* E.g. can be produced by Chinese hamster ovary cells that have integrate cDNA expression vector expressing BiTE protein
37
BiTEs - CD4+ or CD8+ T cells? Discuss
* Although they can exert effects via both CD8+ and CD4+ T cells, CD8+ T cells have been shown to reach maximal tumour cytotoxicity faster (development of a bit exerting only a CD8+ antitumour response may lead to a faster response to treatment)
38
Bargou et al. (2008)
* showed that injection of blinatumomab, an anti-CD19 BiTE, into 38 non-Hodgkin’s lymphoma patients caused an increase in T cell counts resulting from expansion of TEM cells, where counts of TN and TCM cells remained more or less constant
39
BiTE mechanism of toxicity
* BiTEs have been shown to increase expression **of granzyme B** in both CD8+ and CD4+ cells in response to an EpCAM/CD3-BiTE, although a role for Fas in BiTE cytotoxicity does not seem to have widely investigated
Capable of **bystander lysis** and **serial killing**
40
* **Dreier et al 2003**
* Cohorts of five NOD/SCID immunodeficient mice were inoculated with a CD19+ cell line and also treated with human PBMCs (which were the source of T cells)
* Mice treated on multiple days with blinatumomab at different doses
* We can see that at some of the doses blinatumomab completely prevents tumour formation
* No therapeutic effect seen in the presence of human PBMCs alone, vehible control or a BiTE with different target antigen specificity
* Data such as this paved the way for clinical trial
41
* **Goebeler et al 2016**
* Looked at pts with relapsed/refractory NHL. Blina was administered over 4-8 weeks at different doses.
* Looked at 76 patients
* Of those that received that maximum tolerated dose, the overall object response rate was 69%
* Long term follow up analysis of this trial revealed that, among responders OS was \>50% at 8-10 years
42
Discuss approval and current state of BiTEs
* In 2014 the FDA approved Blincyto (blinatumomab), a **CD19-specific BiTE**, for CD19+ Philadelphia chromosome-negative relapsed and refractor B-ALL, with a further approval for Philadelphia chromosome positive B-ALL in 2017.
* Have accounted for 20% of clinical antibody pipeline, yet this is the only approval – problems with on-target off-tumour effects
43
Compare blin administration to CAR T cells
* In contrast to the CAR T cell therapies, however, blinatumomab does **not** require leukodepleting preconditioning and is administered via continuous infusion at a constant flow rate using an infusion pump for 4 weeks in 6-week cycles; this is due its short half-life of around 2 hours
44
BLAST study
a phase II study of blinatumomab, indicate that of 116 patients enrolled, 53% experienced some degree of neurotoxicity, with 5% experiencing encephalopathy and 1% experiencing aphasia.
45
* **Trinklein, 2019**
* They used **NGS-based antibody discovery in humanised rats**
* Found a novel set of anti-CD3 antibodies that bind to multiple epitopes of CD3 with different binding strengths
* Found that candidates at different ends of the potency spectrum elicit differential cytokine release from human primary T cells but had equivalent levels of tumour cell lysis (potency could vary 1000-fold)
* The BiTEs with low levels of cytokine release were able to maintain tumour lysis activity in a mouse xenograft model
* Possible uncoupling of tumour cell killing from cytokine release
* Couldn't provide an accurate assessment of serum cytokine activity in vivo as only supplied mice with small number of human PBMCs
46
When was the first BiTE approved and what for? Why was it recalled?
he first BiTE was approved in 2009 for malignant ascites which was withdrawn due to off-target toxicity
47
What is a major limitation BiTEs when it comes to the treatment of solid tumours?
short half-life in serum (around 2 hours)
48
Iwahori et al. (2015)
* generated T cells capable of secreting BiTEs (ENG T cells) specific for tumour-associated antigen, EphA2.
* In a human lung cancer xenograft model, the group found that following intravenous injection, eGFP- fire fly luciferase-expressing T cells only expanded when co-injected with EphA2-ENG T cells, demonstrating that these ENG T cells can induce expansion of bystander T cells.
They furthered this by showing, following eGFP- fire fly luciferase-expressing T cell injections in human glioma and lung cancer xenograft models, that mice treated with EphA2-ENG T cells had a 2 log or greater reduction in tumour size (compared to CD19-ENG T cells), and this resulted in a long-term tumour-free survival in 5 of the 8 mice studied
49
Discuss scalability of BiTEs
* Cheaper & easier to administer
* Companies developing machine learning prediction platforms that enable well-behaved bispecifics to be predicted from mAbs consistently
50
* **Stone et al (2012)**
* constructed CAR T cells and BiTEs with the same anti-tumour antigen scFv, directed at tumour antibody 237, a murine fibrosarcoma epitope.
* They found that CAR-targetted T cells were more sensitive than BiTE-targetted T cells to low numbers of 237 epitopes per cell.
* To do this, they used Jurkat (~770 epitopes/cell) and JOE (~9,900 epitopes/cells) cells to present the tumour antigens; they found that the JOE line was able to stimulate IFNg release from both CD8+ and CD4+ CAR T cells, whereas neither JOE nor Jurkat lines were able to stimulate CD8+ or CD4+ T cells in the presence of the 237 BiTE.
* It was also found that BiTE-targetted cells were observed to mediate lower levels of Jurkat cell lysis, measured by 51Cr-release assay22.
51
* **Hoseini et al (2017)**
* contrasted these results when comparing a bispecific antibody, BC119, directed at CD3 and GD2 (a target expressed in neuroblastoma and many other tumours) and a CAR T cell with the same anti-GD2 scFv.
* A key observation of this paper was their *in vivo* experiment, using a subcutaneous xenograft of melanoma line M14luc in immunodeficient DKO mice. T cells were injected on days 7, 14 and 21 after tumour initiation.
* It was found that untransduced T cells and BC119 had no effect on tumour size when administered separately, but when administered together caused a rapid reduction in tumour size.
* In contrast, following administration of CAR T cells, a steep initial increase in growth was observed in comparison with BC119, followed by tumour regression with a longer follow-up.
* It was further found via flow cytometry, following subsequent killing of the mice, that the percentage of tumour infiltrating T lymphocytes was higher in the BC119-treated group than the CAR T cell-treated group, although this difference is not significant (P=0.082)
* predominated.
* Suggest that in vivo, BiTEs confer advantage over CAR T cells in redirected T cells to tumours
* Caveats
* the difference observed in tumour infiltrating lymphocytes was not statistically significant
* such xenograft models confer a poor representation of tumour microenvironment and metastasis of respective human tumours
* these results may be specific to the particular melanoma model or mouse line
52
Which are antibody based and which are TCR-based? Discuss difference in terms of specificity
* Antibody-based immunotherapies → CAR T cells and BiTEs
* TCR-based immunotherapies arev → TCR-transduced T cells and ImmTAC molecules
* For all immunotherapies that rely on an antibody targeted recognition of tumour cells to activate a T cell response, perhaps the biggest drawback is **the limited number of surface-presented antigens**, and esp those that are exclusively or largely restricted to tumour tissue - this small number of targets will likely constrain the broad application of these therapies
* BiTEs and CAR T cells are both antibody-based
* ImmTACs recognise tumours from a precisely engineered TCR. They then have an anti-CD3 scFv that engages T cells for effector function – leads to formation of a functional immunological synapse
* These T cells can have any specificity
* Major advantage of these over antibodies – TCRs can target intracellular antigens through recognition of peptide -HLA antigen complexes on the cell surface
* ImmTAC molecules have access to **\>10-fold more** potential cancer targets than current soluble antibody-based bispecific molecules – broadening opportunities across haemaological malignancies and solid tumours
* However, this means they are **HLA-dependent, while BiTEs are HLA independent**
53
What have been some challenges with ImmTACs?
* TCRs are membrane bound would need to be produced as soluble molecules
* Need to increase the affinity of the TCR for its pMHC target
* A high affinity soluble TCR on its own could target a cancer cell but not kill it TCR needs to be fused to an effector molecule
54
Middleton 2019
* Looked at 84 metastatic melanoma pts – all received IMCgp100
* Phase II trial
* In both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma 1 year survival rate was 65%
* This is a **SOLID TUMOUR** – good
* They also found that it increase serum CXCL10 (a T cell attractant) and an increase in cytotoxic T cells in the tumour microenvironment
* This increase in serum CXCL10 or the appearance of a rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival
* _However, efficacy is low – 8.7 %_
* Shows it made the TME inflammatory – could this be used in combination with other immunotherapies??
55
Side effects of ImmTACs
* Concern because of high potency and specificity for target antigen
* Lack of animal models for ImmTAC testing due to differences between human immune system and other species
* Only 40% of MHCI peptidome is shared with humans so any murine surrogate ImmTAC would differ in specificity and binding profile to human TCR
56
Discuss ImmTAC applicability and scale up
* HLA restriction due to the TCR scaffold limits off-the-shelf potentil
* All TCR-based therapies reliant on binding a pHLA complex are restricted to a single or small number of HLA allelic variants, limiting relevant patient populations by HLA allele expression. For example, a common target is the HLA-A\*02:01 allele due to its high representation, yet even this only reaches a frequency of approx. 45%, and only in Caucasian populations
57
Discuss TCR transduced T cell
* Type of adoptive therapy
* T cells are extract by leukapheresis, engineer these to express **a tumour-targetting TCR**, expand these cells and reinject back into the patient
* Will have both genetically modified and endogenous TCR expressed on T cells
* As ImmTACs are, these are also engineered to enhance affinity for tumour Ag
* Require **lower amounts of antigen than CAR** – possibly related ot their involvement of the CD8 or CD4 co-receptors
* **Have recently show to induce responses in solid tumours in addition to synovial sarcoma**
58
Raman, 2016
* demonstrated this was due to cross reactivity with an epitope from the **Titin** protein presented on cardiac tissue despite extensive pre-clinical testing that gave no suggestion of off-target activity
59
Conclusions
* ImmTACs – potential is as readily accessible, potent, cheap and scalable treatment, overcomes major hurdles to widespread CAR-T therapy
* CAR-T – allogeneic transfer feels some distance away – usual immunosuppressive programs given after transplant would defeat the object of reducing tumour immune evasion, thus may need tolerance inducing programs to achieve ‘off the shelf’ use
* CAR-T potential is in sophistication/ precision – could program multiple receptors to program desired effects on metabolism / cell effector programs, all under spatial and temporal control with optogenetics
* BiTEs – development of tri-specific antibody (developed antiCD3xantiCD28xantiCD38 for myeloma developed)
* Use ImmTACs as a temporary boost to immune system while personalised CAR-T are developed?
* Side effects – use sequence based antibody discovery to uncouple cytokine release from anti-tumour activity
* New machine learning and high throughput discovery likely to create many new BiTEs
* *With more combinations of receptors and therapeutic agents, likely to be more side effects and clearly more costs. Argue that should try to optimise existing therapy rather than seeking continuous ‘breakthroughs’*
60
observed that in the pts with CML that relapsed after transplanting you could go back to the original donors and ask them to donate white cells. Infusing these white cells would turn a significant proportion of these patients back into remission
Kolb et al (1990)
61
* construction of first-gen CAR T cells – consist of intracellular domain of CD3z, responsible for ‘signal 1’ of T cell activation
Eshhar et al 1993
62
* Following the construction and antigen stimulation of CD28-CD3z dual-signalling receptors, it was found that these were associated with increased IL-2 secretion as well as increased T cell proliferation with repeated antigen exposure in the absence of any exogenous stimulation, when compared with controls with receptors only expressing CD3z or only CD28
Maher et al 2002
63
* demonstrated in a preclinical model of Raji tumour-bearing immunodeficient mice receiving CAR T cells of distinct T cell subtypes. They found that there was synergistic antitumour activity of CD4+ and CD8+ T cell subtypes in a 1:1 ratio, compared to reduced antitumour activity when either CD8+ or CD4+ were omitted from the formulated product, shown by decreased survival and increased tumour growth seen via bioluminescence imaging
Sommermeyer et al 2016
64
* analysis of 14 patients with B cell malignancies that had been infused with autologous CD19-redirected CAR T cells. They found that the expansion of these cells *in vivo* correlated with the frequency of TSCM cells infused in the first 6 weeks after infusion
* It is thought that naïve T cells (TN) differentiate to form TSCM and TCM cells that are capable of self-renewal, and that these provide populations of TEM and TEFF cells that are incapable of self-renewal, suggesting that the longevity of less differentiated cells provide a greater therapeutic efficacy
Xu et al 2014
65
* The **ELARA trial** of 52 patients with follicular lymphoma showed an overall remission rate of 83% within three months of treatment with Kymirah. However, in this trial, 49% of patients developed cytokine release syndrome (CRS), an adverse side effect consisting of a systemic inflammatory response. This side effect has been responsible for several deaths in similar trials.
* For example, one study was terminated following **death** of 5 patients as a result of fulminant cerebral oedema.
ELARA trial
66
* showed, via multiple independent _single-cell RNA sequencing_ databases and subsequent bioinformatic analysis, that mural cells, which have a critical role in blood-brain-barrier (BBB) integrity, express CD19!
* They confirmed this by **performing IHC** with an anti-human CD19 antibody in healthy subjects post-mortem; CD19 expression was found in cells adjacent to vessel basement membrane walls in perivascular area, along both smaller capillaries and larger vessels.
* They also found that the expression of CD19 in human cells is _significantly higher_ than in murine mural cells, implicating **possible limitations of mouse models** to study immunotherapy-associated toxicity.
* Therefore, a cause of this neurotoxicity may be the targeting of CD19+ mural cells and subsequent BBB disruption.
* However, there are further points which this study might have expanded on; the group have not demonstrated that CAR T cell or BiTE targetting of CD19 in human mural cells is the direct cause of clinical neurotoxicity, and contribution of other processes, such as CRS, may also be important to the pathology.
Avery Posey October 2020
67
* constructed a T cell expressing a combinatorial circuit using a synthetic Notch (SynNotch) receptor, which, following target antigen binding, leads to cleavage of the receptor and release of a transcriptional activator.
* This inducible transcription factor (TF) is able to regulate transcription of a CAR recognising a second tumour antigen.
* Thus, this AND-gate circuit requires binding of antigen to both the SynNotch receptor and the CAR for subsequent T cell activation
* However, they still have little control over these cells after adoptive transfer
* A key observation in this study was their *in vivo* work; they used mice with a **CD19 K562 tumour on one flank** and a **GFP+/CD19+ K562 tumour** on the other.
* They injected T cells bearing an a-GFP synNotch regulating a CD19 CAR and monitored tumour growth.
* They found that in all animals, these cleared the dual-antigen GFP/CD19 tumours but did not clear the CD19+-only tumours which grew at similar rates to negative controls.
* This showed that these T cells would only activate and kill targets in response to multiple tumour antigens, a potential mechanism to spare ‘bystander’ cells expressing single antigens, limiting associated on-target toxicity, such as B cell aplasia or neurotoxicity.
* The study by **Posey’s group** builds upon this, identifying several genes, including *CDJ4**,* *HLA-DRA* and *LTP**,* which are highly enriched on B cells in comparison to brain mural cells which serve as potential targets for combinatorial recognition.
* A limitation of an approach like this may be that both antigens would have to be expressed at considerably high levels by tumour cells.
* Also found that the CAR stays on the surface of the cell for 8 hours – their model showed no cross-reactivity so maybe it is long enough
Roybal 2016
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showed that the ability of these HIF-CAR T cells to kill target cells was significantly improved in hypoxic compared to normoxic conditions; although a caveat of this may be off-target effects in healthy hypoxic tissues, such as the bone marrow.
Juillerat et al. (2017)
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* Current challenge is predicting optimal number of cells to infusion – need better ctrl in space/time
* Off-switches permanently down-regulate activity of CAR T cells
* Developed on-switch CARs which can be activated by light in a confined space
* Cre-loxP expression of CD19-CAR induced by blue light via nuclear translocation and dimerisation of CRY2/CIB1 pair.
* Tested in Jurkat cell line (immortalised T cell line) and primary human T cells, subcutaneous injection into mice - saw long lasting spatial controlled action
* Address duration of light dosing regimens (24hrs in study) + how long CAR activity continued after light stimulus removal
* Blue light doesn’t penetrate tissue as well as red – further develop a system using red, or implantable LEDs
* Induction ability only lasted two days
Huang 2020
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* TRAC T cells
* Most current approach to making CAR T cells insert the CAR-encoding gene into the T cells without disrupting the resident TCR gene
* TRAC T cells – TCR alpha chain constant region
* Used a CRISPR-Cas9 gene editing approach.
* Authors introduced RNA encoding Cas9 into T cells as well as gRNA that targets the TRAC sequence – ds break in TRAC and a viral vectore with a CAR sequence flanked by sequences homologous to the TRAC sequence was introduced into the T cells – original TRAC sequence replaced by CAR sequences
* Deleted the endogenous TCR and replace it with a CAR TCR which was under the control of the promoter for the TCR. Expression regulated in the normal way of a TCR, avoiding overactivation of the T cell.
* They showed this avoided exhaustion in vivo. Used a B-ALL mouse model. Used FACs to determine the number of cells expressing exhaustion markers on day 17 following induction. Found conventional CAR T cells showed up to 50% positive expression for three markers of exhaustion by day 17 whereas less than 2% of the TRAC-CAR T cells did
* Also showed a marked improvement in survival in the mouse model
* Could the integration of viral vectors randomly in the genome be a cause of cancer?
* **Idea for future exp** **** **compare how KOs of components of T cell exhaustion pathways compare with this CAR integration**
* **Eyquem et al (2017)**
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* generation of a switch receptor
* Generated a new chimeric antigen construct in which they linked the PD-1 EC domain to an IC activating domain of CD28 – instead of PD-1 transducing a negative signal, PD-1 transduces an activating signal
* Found that CD19-PD-1/CD28-CAR T cells had superior T-cell proliferation, cytokine production, and sequentially capability of killing PD-L1+ B-cell lymphoma cells *in vitro* and *in vivo* relative to the prototype, CD19-CAR T cells.
Liu et al (2021)
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* designed a CAR targetting tight junction protein claudin-6 (CLDN6), a human tumour-specific antigen.
* They designed a nanoparticulate RNA vaccine, CLDN6-LPX, which promoted CLDN6 expression on the surface of dendritic cells.
* They used mice bearing CLDN6+ lung tumours and administered a subtherapeutic dose of mouse CLDN6 CAR T cells followed by CLDN6-LPX or control.
* It was found that, in the mice receiving CLDN6 CAR T cells and control, tumour growth was only delayed, but the mice receiving CLDN6-LPX demonstrated complete tumour regression and higher median survival.
* Therefore, RNA vaccines could be used clinically to promote CAR T cell expansion for treatment of solid tumours.
Reinhard et al. (2020)
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* Proof-of-concept evidence for allogeneic therapy
* 2 paediatric patients with B-ALL
* Used TALENs to engineer HLA-mismatched donor T cells
* Donor T cells were transduced with lentivirus to express CAR-CD19 and these were electroporated with TALENs to ablate CD52 and T cell receptor- alpha constant region
* CD52 so the cells could evade the conditioning therapy before transplantation and TCR-alpha constant region to minimise graft-vs-host disease
* Molecular remission seen within 1 month in both infants (1 had evidence of skin GVHD, managed with steroids)
* Saw long term remission
Qasim, 2017
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* showed that injection of blinatumomab, an anti-CD19 BiTE, into 38 non-Hodgkin’s lymphoma patients caused an increase in T cell counts resulting from expansion of TEM cells, where counts of TN and TCM cells remained more or less constant
Bargou et al. (2008)
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* Cohorts of five NOD/SCID immunodeficient mice were inoculated with a CD19+ cell line and also treated with human PBMCs (which were the source of T cells)
* Mice treated on multiple days with blinatumomab at different doses
* We can see that at some of the doses blinatumomab completely prevents tumour formation
* No therapeutic effect seen in the presence of human PBMCs alone, vehible control or a BiTE with different target antigen specificity
* Data such as this paved the way for clinical trial
* **Dreier et al 2003**
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* Looked at pts with relapsed/refractory NHL. Blina was administered over 4-8 weeks at different doses.
* Looked at 76 patients
* Of those that received that maximum tolerated dose, the overall object response rate was 69%
* Long term follow up analysis of this trial revealed that, among responders OS was \>50% at 8-10 years
* **Goebeler et al 2016**
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a phase II study of blinatumomab, indicate that of 116 patients enrolled, 53% experienced some degree of neurotoxicity, with 5% experiencing encephalopathy and 1% experiencing aphasia.
BLAST study
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* They used **NGS-based antibody discovery in humanised rats**
* Found a novel set of anti-CD3 antibodies that bind to multiple epitopes of CD3 with different binding strengths
* Found that candidates at different ends of the potency spectrum elicit differential cytokine release from human primary T cells but had equivalent levels of tumour cell lysis (potency could vary 1000-fold)
* The BiTEs with low levels of cytokine release were able to maintain tumour lysis activity in a mouse xenograft model
* Possible uncoupling of tumour cell killing from cytokine release
* Couldn't provide an accurate assessment of serum cytokine activity in vivo as only supplied mice with small number of human PBMCs
* **Trinklein, 2019**
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* generated T cells capable of secreting BiTEs (ENG T cells) specific for tumour-associated antigen, EphA2.
* In a human lung cancer xenograft model, the group found that following intravenous injection, eGFP- fire fly luciferase-expressing T cells only expanded when co-injected with EphA2-ENG T cells, demonstrating that these ENG T cells can induce expansion of bystander T cells.
They furthered this by showing, following eGFP- fire fly luciferase-expressing T cell injections in human glioma and lung cancer xenograft models, that mice treated with EphA2-ENG T cells had a 2 log or greater reduction in tumour size (compared to CD19-ENG T cells), and this resulted in a long-term tumour-free survival in 5 of the 8 mice studied
Iwahori et al. (2015)
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* constructed CAR T cells and BiTEs with the same anti-tumour antigen scFv, directed at tumour antibody 237, a murine fibrosarcoma epitope.
* They found that CAR-targetted T cells were more sensitive than BiTE-targetted T cells to low numbers of 237 epitopes per cell.
* To do this, they used Jurkat (~770 epitopes/cell) and JOE (~9,900 epitopes/cells) cells to present the tumour antigens; they found that the JOE line was able to stimulate IFNg release from both CD8+ and CD4+ CAR T cells, whereas neither JOE nor Jurkat lines were able to stimulate CD8+ or CD4+ T cells in the presence of the 237 BiTE.
* It was also found that BiTE-targetted cells were observed to mediate lower levels of Jurkat cell lysis, measured by 51Cr-release assay22.
* **Stone et al (2012)**
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* contrasted these results when comparing a bispecific antibody, BC119, directed at CD3 and GD2 (a target expressed in neuroblastoma and many other tumours) and a CAR T cell with the same anti-GD2 scFv.
* A key observation of this paper was their *in vivo* experiment, using a subcutaneous xenograft of melanoma line M14luc in immunodeficient DKO mice. T cells were injected on days 7, 14 and 21 after tumour initiation.
* It was found that untransduced T cells and BC119 had no effect on tumour size when administered separately, but when administered together caused a rapid reduction in tumour size.
* In contrast, following administration of CAR T cells, a steep initial increase in growth was observed in comparison with BC119, followed by tumour regression with a longer follow-up.
* It was further found via flow cytometry, following subsequent killing of the mice, that the percentage of tumour infiltrating T lymphocytes was higher in the BC119-treated group than the CAR T cell-treated group, although this difference is not significant (P=0.082)
* predominated.
* Suggest that in vivo, BiTEs confer advantage over CAR T cells in redirected T cells to tumours
* Caveats
* the difference observed in tumour infiltrating lymphocytes was not statistically significant
* such xenograft models confer a poor representation of tumour microenvironment and metastasis of respective human tumours
* these results may be specific to the particular melanoma model or mouse line
* **Hoseini et al (2017)**
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* Looked at 84 metastatic melanoma pts – all received IMCgp100
* Phase II trial
* In both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma 1 year survival rate was 65%
* This is a **SOLID TUMOUR** – good
* They also found that it increase serum CXCL10 (a T cell attractant) and an increase in cytotoxic T cells in the tumour microenvironment
* This increase in serum CXCL10 or the appearance of a rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival
* _However, efficacy is low – 8.7 %_
* Shows it made the TME inflammatory – could this be used in combination with other immunotherapies??
Middleton 2019
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* demonstrated this was due to cross reactivity with an epitope from the **Titin** protein presented on cardiac tissue despite extensive pre-clinical testing that gave no suggestion of off-target activity
Raman, 2016
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* Compared the tumour response of erbB2-specific CAR T cells in the Her2NG transgenic mouse – overexpresses human erbB-2 transgene under MMTV promotor – these mice progressively develop mammary tumours
* They found a single administration of CAR T cells lead to **rejection of the primary tumour**, but a few weeks later tumours relapses in mammary gland and remote sites. This could be controlled by repeated CAR T cell injections and one mice remained cancer free for **500 days** following treatment
* They also found that CAR T cells could be used prophylactically a single injection of CAR T cells before the appearance of any mammary tumours could significantly delay the appearance of mammary tumours for several months
* Could we use them in high-risk patients prophylactically? – probably not (SEs)
Levin 2014
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Discuss recent ImmTAC approval
Jan 2022 FDA approved for metastatic uveal carcinoma
Pts need HLA-A\*02:01 (ony 45% remember)
Shown to sig extend overall survival
Attaches to the HLA-A\*02:01 complex
